Hypoxic pulmonary vasoconstriction (HPV) plays important roles in normal and diseased lungs. Pulmonary arterial smooth muscle cells (PASMCs) contain all essential mechanisms of HPV, which depends on influx of calcium and secondary increases in intracellular calcium concentration. It is generally accepted that this calcium influx occurs through sarcolemmal voltage-dependent calcium channels (VDCCs) activated by depolarization due to hypoxic inhibition of voltage-dependent potassium (Kv) channels. However, observations that HPV was prevented by inhibition of calcium release from sarcoplasmic reticulum (SR), but not depolarization or inhibition of Kv and VDCCs, suggest that mechanisms of HPV are more complicated. Our preliminary data demonstrate that hypoxia enhanced capacitative calcium entry (CCE) through PASMC sarcolemmal channels activated by SR calcium depletion. Inhibition of CCE blocked hypoxia-induced increases in intracellular calcium concentration in PASMCs and HPV in isolated lungs. Distal pulmonary arteries expressed TRPC mRNA and proteins, homologs of "transient receptor potential" proteins in Drosophila that are thought to form CCE channels. On this basis, we hypothesize that HPV is initiated by SR calcium release and secondary activation of CCE through channels composed of TRPC proteins, causing local increases in intracellular calcium concentration. Depolarization occurs next, either directly due to opening of CCE channels or indirectly due to local calcium-dependent alteration of sarcolemmal K or CI channel-activity. The resultant calcium influx through VDCCs augments CCE, raising global intracellular calcium concentration sufficiently to trigger PASMC contraction. To test this hypothesis, we will measure vasomotor responses in isolated lungs and use fluorescent microscopy, patch clamping, and small interfering RNAs in PASMCs to determine how hypoxia releases SR calcium and alters the electro-physiologic characteristics of CCE channels, whether activation of CCE channels causes hypoxic depolarization, how effects of hypoxia on CCE are transduced, and whether CCE channels responsible for hypoxic responses in PASMCs are composed of TRPC proteins. We hope that these experiments will improve understanding of HPV and ultimately lead to decreased morbidity and mortality in patients with hypoxic pulmonary hypertension.